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Thursday, November 13, 2014

Evolution of malaria resistance: 70 years on...and on....and on

It was about 70 years ago that the complex problem of anemia, malaria, and genetic interactions, with their relation to hemoglobin was first beginning to be understood. Sickle cell anemia and its association with a globin gene variant, and similar associations between malarial susceptibility and other genes (such as G6PD and Duffy and other globin gene mutations) were also rapidly identified in roughly the same decades. The findings were showing that in areas of the world with long-endemic malaria, various gene mutations seemed to be at high frequency as if they protected against malaria. I was never involved in this directly, but I studied under Frank Livingstone and James V Neel at Michigan, two of the leaders in understanding the evolution of the protective mechanisms.

For decades we have had direct clinical evidence, mainly in Africa, but also in Sardinia, and then later in other places including southeast Asia, that at least some of the putatively protective mutations in the alpha and beta globin, and other genes did in fact protect against malaria, but that they had side effects such as various forms of anemia or other problems. Even then most of the evidence was circumstantial and based on geographic correlations.

The idea of a balanced polymorphism was suggested in regard to these variants. If you had two 'malaria-protective' alleles at the gene (one in each copy of the gene that you have), you were vulnerable to anemia, but if you had two 'normal' alleles you were susceptible to malaria; however, having one of each (a heterozygote genotype) you had some protection against both malaria and anemia. Evolution favored keeping both variants in the population, because selection worked against both homozygotes.

Far beyond malaria: Relationship to fundamental evolutionary questions
The idea of balanced polymorphisms played into a major theoretical argument among evolutionary biologists at the time, and sickle cell anemia became a central case in point, and a stereotypical classroom example. But the broader question was quite central to evolutionary theory. Balancing selection was, for many biologists who held a strongly selectionist version of Darwinism, the explanation for why there was so much apparently standing genetic variation in humans, but generally in all, species.

The theory had been that harmful mutations (the majority) are quickly purged, so the finding that there was widespread variation (polymorphism) in nature at gene after gene, the result of the type of genotyping possible then (based on protein variation), demanded explanation; balanced polymorphism provided it. This was countered by a largely new, opposing view called 'non-Darwinian' evolution, or the 'neutral' theory; it held that much or even most genetic variation had no effect on reproductive success, and the frequency of such variants changed over time by chance alone, that is, experience 'genetic drift'. This seemed heretically anti-Darwinian, though that was a wrong reaction and only the most recalcitrant or rabid Darwinist today denies that much of observed genomic variation evolves basically neutrally. But many saw the frequency of variants associated with what were seen as serious recessive diseases,
like PKU and Cystic Fibrosis (and others) as the result of
balancing selection.

In support of the selectionist view, many variants have been found in the globin and other genes for which the frequency of one or more alleles is correlated geographically with the presence (today, at least) of endemic malaria. But there are lots of variants that might be correlated with other things geographic because the latter are themselves often correlated with population history. Thus, the correlations are often empirical but not clearly causal. Indeed, not many variants have been clearly shown experimentally or clinically actually to be functionally related to malaria resistance.

In this light it is interesting to see a rather large-scale attempt at testing whether putative malaria-associated variants really are protective. The paper ("Reappraisal of known malaria resistance loci in a large multi center study") by a large consortium of authors is in the November 2014 Nature Genetics; it is paywalled so if you don't have direct access but would like to read it, I'd be happy to email a pdf.

These authors compiled large data sets from different areas of the world which have endemic malaria caused by the specific falciparum subtype of parasite, and compared the frequency of the many candidate gene variants in sufferers of severe malaria to a large set of unaffected controls (of course some of them may later become affected).

A long time coming...and the clock still ticking
Even now, 70 years after the first ideas were suggested, we still have scant direct clinical data showing protection at a mechanistic level, so the results of this paper are still statistical. But they are at least from a reasonably designed and specific study. The authors found positive statistical association for some of the most clear-cut classical risk alleles (sickle cell, G6PD, O-blood group), but ambiguous or variable evidence even for some of these, and no statistical evidence for many other putative causal, or protective variants. Further, they found that some variants had different effects in males and females, and one SNP, in the CD40LG gene, previously found to be associated with severe malaria, was associated with reduced risk in The Gambia, but significantly increased risk in Kenya. Whether this is just statistical variation or indicators of other aspects of these local-area genomes isn't clear.

The evidence in the positive instances is persuasive, even if just statistical, but the conflicting results and the surprising lack of findings for so many is curious as well as discouraging. How can it be that so long on, we still basically don't even know if a genetic variant is protective or not, other than the most classical ones? This shows how very challenging even 'simple' causation can be.

This raises the basic evolutionary issue in a different way. Darwin was convinced that adaptive evolution was very slow. One major reason was that rapid changes of species or adaptations were rarely observed (still true), and if they occurred they could be interpreted as creationist rather than natural events. Adaptive evolution under human direction, as in agricultural breeding, clearly brings about easily measured change. But some forms of natural selection could be quite strong. Adaptive coloration is one, but malaria should be another because it is so common and strong a negative effect on health. So basic evolutionary arguments ought, it was long hoped, demonstrate that, in this instance, balancing selection was a correct explanation of at least these polymorphisms.

In past work, one hemoglobin variant (called hemoglobin E) has apparently been sweeping across southeast Asia because there was no down side to being an EE homozygote, and it protected against malaria. But generally, the actual selective effect has been very hard to prove. The new study shows this in a sobering way. Is the story right? Have prior speculations about protective mutations been too superficially offered, and incorrect? Is the selective effect so small even in relation to malaria, that we can't see it even with samples large enough that 'nature' could have made a detectable selective difference? Or, if so gradual in a Darwinian sense, do these other mutations really make an evolutionary difference?

Several relevant points are, first, that this study only looked at one form of malaria (P. falciparum), and second, that the different putative protective genes are involved in different physiological pathways. And, as even the authors note, current patterns of disease, when antimalarial drugs are widely used, may not reflect patterns in the past, and thus it may not be possible to conclude that P. falciparum was the selective force these results suggest it may have been, plausible though that seems to be. These points suggest that even here, complexity and subtlety are involved.

Beyond evolutionary theory
More sobering than the reality of detecting evolutionary or even genuine physiological differences among these various genotypes, is the further fact that even for these major and rather clear causal sites, there is still basically no progress in effective gene-based therapy. After all, the target cells are in blood (generally, red cells), among the most easily accessible of all tissues. Given the unrestrained promises repeatedly being made by the genomewide-do-everything industry, this is (or should be) a very sobering thought. Our technological tools should, one might expect, have been able to solve such comparatively clear-cut problems.

To us, this 'failure' indicates the subtlety of genome physiology. Given the hundreds of putatively causal single-gene findings by GWAS and other means, where the evidence has seemed strong, we should be showing that genomic data are, after all the expense and effort, really worth gathering. We should be making a definitive, and one might say systematic, march toward elimination of these genetic threats, perhaps the way vaccines have done against many infectious diseases. If we could actually do that, and speak of cures and prevention rather than just risk-estimation of countless minor factors, then nobody would disagree that further genomic big-science efforts were worth the investment.

Meanwhile, more than 70 years on, the largely failed effort to use that knowledge directly to rid our species of a disease that has been estimated to have killed more human beings than any other single cause, shows how far we have to go--and how important new sorts of thinking could potentially be to the effort.

And, into the bargain, perhaps we're learning a lot about how adaptive evolution works, reinforcing Darwin's ideas about its slowness, about multiple alternative or interactive pathways, and more.

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